The long term goal of this project is to characterize pre-synaptic mechanisms regulating synaptic transmission, with a focus on G protein pathways. To maintain a stable pattern of synaptic transmission, the rates of SV exocytosis and endocytosis must remain in balance. Thus, G protein pathways that modulate synaptic transmission must produce coordinated changes in SV exocytosis and endocytosis. Here we propose to continue this project by analyzing how exocytosis and endocytosis are regulated. RAB-3 is a small GTPase that undergoes a cycle of association and dissociation with synaptic vesicles (SVs) in a manner that depends upon its bound guanine nucleotide. Prior studies showed that RAB-3 regulates SV docking, and the probability that an SV will fuse following depolarization. Despite its impact on synaptic transmission, relatively little is known about how RAB-3 is regulated. In the last funding period, we identified three new regulators of RAB-3, and showed that RAB-3 promotes both SV and dense core vesicle (DCV) secretion. We also initiated a new project characterizing the mechanism by which Endophilin, a conserved membrane associated protein, promotes SV endocytosis. Based on these preliminary data, we propose three new Aims. First, we will determine the mechanism by which Synaptobrevin and EGL-30 G1q regulate RAB-3. Second, we will screen for new regulators of RAB-3. Third, we will test the importance of Endophilin's two functional domains (the membrane binding N-Bar domain, and the SH3 domain). These experiments will allow us to distinguish between two competing models for Endophilin function. We will also determine how Endophilin is regulated by exocytosis. In summary, these Aims address two interesting aspects of presynaptic cell biology. The ability of synapses to operate over a wide range of signaling rates is dependent upon efficient coordination of the SV cycle of exo- and endocytosis. RAB-3 promotes SV fusion, and its regulation could provide a simple biochemical mechanism for producing synaptic plasticity. The experiments proposed here should give significant new insights into how RAB-3 is regulated, and how its activity is coupled to the SV cycle. Endophilin is required for SV endocytosis, and our preliminary results suggest that its availability at synapses is regulated by the rate of SV exocytosis. Thus, our analysis of Endophilin is likely to provide insights into how these two processes are coordinated. Since RAB-3 and Endophilin play analogous roles in mammalian synaptic transmission, it is likely that our results will also provide new insights into presynaptic mechanisms in mammals. PUBLIC HEALTH RELEVANCE: This proposal describes a coherent set of genetic, biochemical, and biophysical experiments designed to characterize pre-synaptic mechanisms regulating synaptic transmission. In particular, we propose to characterize how two Rab proteins (Rab3 and Rab27) are regulated, and how their activities are coupled to the synaptic vesicle cycle. We also will characterize the mechanism by which Endophilin promotes synaptic vesicle endocytosis.